The Experiment Study On The Immunogenicity Of Nogo-66 And The Immunoprotection To RGCs In SD Rats With Chronic Ocular Hypertension

The character of damage in glaucoma show strong resemblance to degenerated central nervous system (CNS). Just a few damaged retinal ganglion cells(RGCs) will trigger the self-propagating chain of hazard events and lead to the injury of vicinity nerve cells.As the immune privilege of CNS, the primary degeneration will result in cytotoxicity of internal environment. It is mean, the degenerated nerve cells initiate a series of secondary degeneration (SD) and the latter trigger more wider-bound and deeper layer injury. So it seems that the SD means the injuryed nerve cells intiate the autoimmune response and the autoimmune response intiate turn into futhermore injury. Even if the classic ways in clinical, we have no other achievement but remove the intraocular hypertension of glaucoma ,it still on its way of SD ahead come from the degeneration of RGCs leads to special cytotoxicity microenvironment.Bakalash(2003)found that immunized Rats with high IOP-induced RGC loss was reduced by vaccination with R16(a peptide derived from interphotoreceptor retinoid-binding protein) and the benefit of protection against IOP-induced RGC loss outweighed the cost of the monophasic experimental autoimmune uveitis (EAU) that transiently developed in a susceptible rat strain.There are lots of factors act in the secondary degeneration coming from nervous degeneration . the most significance one is myelin inhibitor Nogo which express Nogo-A, Nogo-B and Nogo-C whose common Nogo-66 topology outside cellular membrane. In CNS post-trauma, Nogo-66 present and bind NgR leading to inhibiting result in nerve cells'survival. It has been named as self-propagating chain( SPC) . As a immune privilege apparatus, the CNS can not identify Nogo-66 as a damaging antigen which result in antigenic specificity T lymph cell keep unreactiveness. No available intervention is practiced to break the SPC which lead a infernal circle between secondary damage and damaging antigen. Our study on immunoprotection in nervous damage has discovered that Nogo was widely expressed during chronic ocular hypertension in retina and according to the level of ocular hypertension. Accordingly to these results, we designed to immune the injuried RGCs with exogenous Nogo-66 that we got by genetic recombination owing the same amino acid array with the endogenous ones. So we get the Nogo-66 special T lymph cells activation and T cell mediated autoimmune responce that break the injuring SPC and achieve effective immunoprotection to damaged RGCs.We have got T lymph cell epitope in Nogo-66 through ProPred MHC Class-II Binding Peptide Prediction Server software by ProPred web interface procedure. Coming from achieved work, What we do in this study lately as follow: 1. Characterizated the OX40L expressing on microglia and the volume of every group cells under the in different level of protein Nogo-66 with immunocytochemistry (ICC),contrasting with the normal and the different strength of protein Nogo-66 cell culture fluid by different density. 2. To study the influence of Nogo-66 to the retinal immunologic character of SD rats by immunising the experimental animals by Nogo-66 protein and detect IgG in blood serum for verifying the change in humoral-mediated immunity,detecting the percentage of T cell in S and G2/M stage to know the change about potential improving of cell mediated immunity at the pre-immunse and post-immunised different time points. While the experimental autoimmune encephalomyelitis( EAE) and experimental autoimmune uveitis( EAU) was detected post the vaccinated with Nogo-66 of different level of density. 3. The study about the character of immune in retinal microglia during chronic ocular hypertension in SD rats:Building the animal model of chronic ocular hypertension by 532-laser light coaglation and detected the CD11b and MHC-Ⅱexpression in the retinal's microglia by immuno- histochemistry , Dextran Tetramethyl Rhodamine (DTR ) labeled the RGCs and amounted them to comparison the experiment and control groups. 4. The study on the immune character of retinal microglia and immunoprotection to RGCs in SD rats with chronic ocular hypertension. Based on those immune character of Protein Nogo-66, injection were acted to the chronic intraocular hypertension SD rats as animal model by 532-laser. Then we detected the CD11b expression in the retinal's microglia by immunohistochemistry , Dextran Tetramethyl Rhodamine (DTR ) labeled the RGCs and amounted them. To contrasted the amount of microglia cell for phagocytose and the residual RGCs between control and experiment group.The main results and conclusions are as follows:1. Comparing with the control group, retinal microglia expressing more significant level charged by protein Nogo-66. the activated microglia showed bigger volume and less cell process ,contrasting the different experiment groups. The megalophage( MP) like cells appeared when concentration of Nogo-66 arrived 2000ng/ml. meanwhile, retinal microglia could be activated by protein Nogo-66 and expressing higher level OX40L,maybe contribution on the immune situation.2. The immunition of Nogo-66 protein improving both the humoral- mediated immunity and cell-mediated immunity be proved by in vivo. The immuned SD rats IgG show higher concentration in serum and T cells of S stage in splenic lymphocyte ( SPL) than control groups. Meanwhile, under the different concentration of Nogo-66, no experimental autoimmune encephalomyelitis( EAE) or experimental autoimmune uveitis( EAU) occurrenced.3. Retinal microglia get actived function of swallow while lost the function of antigen presention under the chronic ocular hypertension. As ocular hypertension continuing, retinal microglia expressed adhesion molecule CD11b showing phagocytosis action. Whilemean, through the process of ocular hypertension, retinal microglia expressed no MHC-Ⅱthat maybe come from immune privilege CNS.4. The immunoprotection of Nogo-66 to RGCs in SD rats with chronic ocular hypertension. Vaccinating the Nogo-66 to SD rats 7d before the ocular hypertension( OHT), and strengthen it 1m after the OHT by 100μg,We got retinal microglia charactered with expressed MHC-Ⅱand less expressed CD11b in internal granular layer(IGL) and inner plexiform layer(IPL), as the stronger expressed CD11b in the nerve fiber layer (NFL). The vaccination show immunoprotection to RGCs , which verified by the amount of RGCs and LP1 as well as AP1 of FVEP 2m after OHT.